Download Antidiarrheal activity of methanol leaf extract of Lophira Lanceolata

Merit Research Journal of Environmental Science and Toxicology (ISSN: 2350-2266) Vol. 3(4) pp. 059-064, April, 2015
Available online http://www.meritresearchjournals.org/est/index.htm
Copyright © 2015 Merit Research Journals
Original Research Article
Antidiarrheal activity of methanol leaf extract of Lophira
Lanceolata Tiegh (Ochnaeceae)
Nneka Igboeli1, Collins A. Onyeto*2, Augustine N. Okorie2, Florence N. Mbaoji2,
Ifeoma A. Nwabunike2 and Daniel I. Alagboso1
Abstract
1
Department of Clinical Pharmacy,
University of Nigeria, Nsukka, 410001
Enugu state, Nigeria
2
Department of Pharmacology and
Toxicology, University of Nigeria,
Nsukka, 410001 Enugu state, Nigeria
*Corresponding Author’s E-mail:
[email protected]
Tel: +2348034004221
This study investigated the effect of methanol leaf extract of Lophira
lanceolata for antidiarrheal and spasmolytic properties in experimental
albino Wistar rats. The methanol extract (ME) obtained by 72 h maceration
was evaluated for antidiarrheal activity using Castor oil induced diarrhea,
GIT transit of Charcoal meal model in rodents and isolated guinea pig ileum
model. The extract exhibited antidiarrheal and antispasmodic activity in a
non-dose dependent manner. At 100 mg/kg, extract produced highest
percentage diarrhea protection (63.33%) when compared to the negative
control while 79.95% protection was recorded with loperamide. The extract
exhibited highest anti-motility effect at 200 mg/kg of the extract (giving
5.14% motility inhibition as against 31.34% of atropine). The extract at 500µg
completely blocked contraction induced by 5µg of acetylcholine. Similarly,
1µg of Atropine also blocked contraction induced by 5µg of acetylcholine.
The results showed that the methanol leaf extract of Lophira lanceolata has
antidiarrheal and antispasmodic activity which supports its folkloric
medicinal use.
Keywords: Acetylcholine, Antidiarrheal, Antispasmodics, Guinea pig, Lophira
lanceolata.
INTRODUCTION
Diarrhea is an increase in the frequency of bowel
movements or a decrease in the form of stool or change
in the consistency of stool (greater looseness of stool). It
can also be defined as an increase in the volume of stool
or frequency of defecation. World Health Organization
(WHO) defined diarrhea as three or more watery or loose
bowel movements in a 24 hour period (WHO, 2014).
Diarrhea is one of the most common clinical signs of
gastrointestinal disease, but also can reflect primary
disorders outside of the digestive system (Christian,
2014).
Worldwide, diarrhea claims several million lives
annually, mostly those of infants. In 2004 approximately
2.5 billion cases of diarrhea occurred worldwide
which results in 1.5 million deaths among children
under the age of five. Greater than half of these were in
Africa and South Asia. Diarrhea remains the
second leading cause of infant mortality (16%)
after pneumonia (17%) in this age group (Teke et al.;
2003).
Lophira lanceolata among other folkloric medicinal
uses has been used to treat diarrhea, dysentery,
menstrual pain. However, these claims are yet to be
studied.
The goal of this study was to investigate the Lophira
lanceolata for antidiarrheal activities.
060 Merit Res. J. Environ. Sci. Toxicol.
MATERIALS AND METHODS
Animals
Male Guinea pig (350g) and Wistar albino rats (115210g) of either sex were obtained and housed in cages,
acclimatized under normal laboratory environmental
conditions and feed in the animal house facility of the
Animal House of the Department of Pharmacology and
Toxicology, University of Nigeria, Nsukka. The animals
were maintained freely on standard pellets and water. All
animal experiments were in compliance with the
National Institute of Health Guide for Care and
Use of Laboratory Animals (Pub No. 85 – 23, revised
1985).
Plant Collection, Authentication and Preparation
The fresh leaves of Lophira lanceolata was collected from
Enugu state, Nigeria in August, 2013. The plant was
identified and authenticated by Mr. A Ozioko, a
Taxonomist and staff of the International Centre for Ethno
medicine and Drug Development (InterCEDD) Nsukka,
Nigeria. The fresh leaves were air-dried at room
temperature properly and then grounded into
powder using a grinder and weighed using a weighing
balance.
Extraction Procedure
About 5kg of the powdered material was macerated with
20 L of 80% methanol for 72 h with constant shaking;
the resultant mixture was filtered using a nylon cloth and
the filtrate concentrated to dryness at room
temperature. The extract was stored in a freezer at
o
-15 C, required concentrations were reconstituted as
needed.
Determination of Percentage extractive Yield
Percentage yield was calculated as the weight of the
filtrate divided by the total weight of the ground powder in
percentage.
Yield (%) = [wt of extract (g)/wt of plant material (g)] x
100.
Chemicals, solvents and reagents
Methanol (Sigma Aldrich, Germany), Tween 80, distilled
water, Charcoal, tragacanth, NaCl, KCl, MgCl2, NaHCO3,
NaH2PO4, Glucose, CaCl2. Loperamide (Drugfield
Nigeria), Castor oil, atropine sulphate, Acetylcholine,
Histamine, and Promethazine.
Equipment
Analytical weighing balance (Furi; FEJ-600, China),
kymograph (Ugo Basile, Germany), tissue organ bath
(Ugo Basile, Germany).
Pharmacological Tests
Castor oil-induced diarrhea in rats
Thirty Swiss albino rats of either sex were randomly
selected into 5 groups (n=6) and fasted for 18 hours
(Sunil et al.; 2001).
Group I (negative group) received 2ml/kg of distilled
water, Groups II,III,IV received 100,200, and 400mg/kg of
plant extract respectively while group V (positive group)
received Loperamide (4m/kg) per orally. After 1hour of
drug treatment, 2 ml of Castor oil was given orally to each
rat. The onset time was noted and diarrhea defecations
were observed hourly for 6hours. Inhibition of defecation
(%) was calculated.
Inhibition of defecation (%) =
Where Mc: mean defecation caused by castor oil, Md:
mean defecation caused by drug or extract (Williamson,
1996)).
Gastrointestinal Transit Time in rats
Experimental rats of either sex (115-150g) grouped
randomly into 5 groups of (n=6) and fasted for 18hours
(Sunil et al.; 2001).
Group I (negative group) received 2ml/kg of distilled
water, Groups II, III, IV received 100, 200, and 400mg/kg
of plant extract orally while group V (positive group) was
given 4mg/kg of atropine orally. After 1hour of drug
administration, each animal was given 2ml of standard
charcoal meal (10% activated charcoal suspension in
10% tragacanth gum).
The rats were sacrificed 1hour after the administration
of the charcoal meal, the abdomen were opened and the
small intestine was immediately isolated. The length of
the intestine from pylorus to the caecum (LSI)
and the distance travelled by charcoal (LM) were
measured.
The peristaltic index (PI) and % inhibition
for each rat were calculated using relations below;
Igboeli et al. 061
;
PI=Peristaltic Index; LM=Length
LSI=Length of Small Intestine.
Mean + SEM.
;
Charcoal
Meal;
In vitro testing on Guinea pig Ileum
The
isolated
tissue
preparations
according
to the technique of Perry (Perry, 1982) and
Williamson et al. (de la Puerta and Herrera 1995) were
employed.
A male guinea pig (350g) fasted for 18h, was
sacrificed to obtain a 2cm long pieces of ileum for the
experiment. A piece of guinea pig ileum was tied with the
aid of
thread at both ends and suspended in a
0
thermoregulated 50 ml organ bath, maintained at 37 C,
containing a Tyrode solution. (Galvez, 1996; Onyeto et
al., 2014).
The suspended ileum was allowed to equilibrate for 45
minutes and the solution was refreshed every 15 minutes
with Tyrode solution. After the initial equilibration
period, acetylcholine (5µg) then followed by histamine
(5µg) as controls. The methanol extract of Lophira
lanceolata at organ bath concentrations of 1µg/ml,
10µg/ml, 1000µg/ml, 2000µg/ml and 4000µg/ml were
added alone for effects, then in co-administration with
acetylcholine (5µg) and histamine (5µg) respectively until
complete blockade was observed in each case. The dose
of the plant extract that completely blocked
acetylcholine (5µg) and histamine (5µg) were noted.
Then varied doses of promethazine and atropine were
then added with corresponding administration of
histamine (5µg) and acetylcholine (5µg) respectively and
the point of complete blockades were noted
respectively.
Isometric contractions were recorded on kymograph
paper. Relaxation (%) was calculated using the relation;
RESULTS
Percentage Extractive Yield
The extraction process afforded 331 g (6.55% w/w) of the
methanol extract.
Phytochemical Constituents of Extract
The methanol leaf extract of Lophira lanceolata gave
positive test for flavonoid, alkaloid, glycoside, saponins,
and terpenoids, reducing sugar, oils and carbohydrates
(Tunaru, 2012).
Pharmacological Tests
Acute Toxicity Test
Oral administration of methanol extract of L.lanceolata up
to 5 g/kg caused no death in mice. Therefore, the oral
LD50 of the aqueous extract in mice was >5 k/kg. Also
there were no signs of obvious behavioral and physical
adverse effects (Tunaru, 2012).
Effect of Extract on Castor oil-induced diarrhea in
rats
The extract inhibited castor oil-induced diarrhea in nondose dependent manner with highest inhibition
exhibited at 100mg/kg. The degree of inhibition
was
expressed
as
percentage
inhibition
of
defecation (Table 1).
Relaxation (%) =
Where Ho=Original response height, Hr=height of
relaxation response.
The antihistamine effects of the ME extract at different
doses were compared with promethazine (1µg and 5µg)
and the anticholinergic effects of the ME extract at
different doses were equally compared with atropine (1µg
and 5µg).
Effect of Extract on GIT Transit Time of Charcoal
meal
The extract caused a non-dose dependent decrease in
the movement of charcoal meal in rats. The degree of
inhibition of the peristaltic movement expressed as
percentage inhibition of movement was highest at
200mg/kg dose of extract (Table 2).
STATISTICAL ANALYSIS
Results obtained were subjected to one-way analysis of
variance
(ANOVA)
using
Graph
pad
Prism
Version 5, followed by Dunnets post hoc test and P<0.05
was considered significant. The results are expressed as
Effect of Extract on guinea pig ileum
Atropine and promethazine at 1µg and 5µg respectively inhibited 5µg of acetylcholine and 5µg histamine
062 Merit Res. J. Environ. Sci. Toxicol.
Table 1. Effect of Extract on Castor oil-induced diarrhea in rats
Groups
I
II
III
IV
V
Treatment
Control
ME
Loperamide
Dose mg/kg
2ml
100
200
400
4
Mean defecation in 6 hours ± SEM
0.8333±0.3752
0.1667±0.1361
0.3056±0.2412
0.4722±0.2667
0.1111±0.1111
%Inhibition of defecation
0.00
79.95
63.33
43.34
86.67
ME, Methanol extract. The values are mean ± SEM., n=6, *P<0.05 vs control.
Table 2. Effect of Extract on GIT Transit Time of Charcoal meal
Groups
I
II
III
IV
V
Treatment
Control
ME
Atropine
Dose
mg/kg
2ml
100
200
400
3
Length of Intestine ±
SEM(cm)
98.0000±2.6330
105.0000±2.1910
97.3300±3.0070
103.8000±3.7600
101.7000±0.6667
Movement Of
Charcoal ± SEM(cm)
98.000±2.633
100.800±2.713
92.330±5.220
102.400±4.007
69.830±15.110*
%Inhibition of
movement
0.00
4.00
5.14
1.35
31.34
ME, Methanol extract. The values are mean ± SEM., n=6, *P<0.05 vs control.
Table 3. Result of Responses of extract on acetylcholine induced contractions
Dose
5µg Ach
5µg Ach
Mean
5µg Ach+10µg ME
5µg Ach+50µg MEL
5µg Ach+100µg MEL
5µg Ach+500µg MEL
5µg Ach+1mg MEL
5µg Ach+1µg Atropine
5µg Ach+5µg Atropine
Height of Response(cm)
4.20
4.00
4.10
0.40
0.30
0.70
0.00
0.00
0.00
0.00
% Relaxation
90.00
92.00
82.93
100.00
100.00
100.00
100.00
Response
Contraction
Contraction
Relaxation
Relaxation
Relaxation
Blocked
Blocked
Blocked
Blocked
ME, Methanol extract. The values are mean ± SEM., n=6, *P<0.05 vs control.
Table 4. Result of Responses of extract on Ach induced contractions
Dose
5µg Histamine
5µg Histamine
Mean
5µg Hist+10µg ME
5µg Hist+50µg ME
5µg Hist+100µg ME
5µg Hist+500µg ME
5µg Hist+1mg ME
5µg Hist+5mg ME
5µg Hist+10mg ME
5µg Hist+50mg ME
5µg Hist+100mg ME
5µg Hist+1µg Promethazine
5µg Hist+5µg Promethazine
Height of Response(cm)
3.30
3.50
3.40
3.70
3.30
3.20
3.00
2.60
2.50
1.70
0.50
0.00
0.30
0.00
% Relaxation
0.00
2.94
5.88
11.76
23.53
26.47
50.00
85.29
100.00
91.18
100.00
ME, Methanol extract. The values are mean ± SEM., n=6, *P<0.05 vs control.
Response
Contraction
Contraction
Contraction
Relaxation
Relaxation
Relaxation
Relaxation
Relaxation
Relaxation
Relaxation
Blocked
Relaxation
Blocked
Igboeli et al. 063
contractions respectively while 500µg and 100mg of
extract caused complete blockade of 5µg of Acetylcholine
and 5µg histamine contractions respectively (Tables 3
and 4). At 1µg and 5µg doses of atropine and
promethazine respectively, there were inhibitions of
concentrations produced by acetylcholine and histamine
each at 5µg whereas there was a complete blockade of
the agonistic effects of both drugs at same dose as
above by 500µg and 100µg of the extract respectively.
DISCUSSION
Phytochemical studies of methanol extract of leaves of
Lophira lanceolata revealed the presence of abundance
of flavonoids. Alkaloids, glycosides and oil were
moderately present while saponins, terpenes, reducing
sugars, carbohydrate and acidic compounds occurred in
relatively small quantities (Tunaru, 2012).
Acute toxicity test suggests a considerable good
safety profile such that even 5000mg/kg/day oral dose, of
the leaf extract is still safe for experimental rats as
reported by Onyeto et al (Tunaru, 2012).
Eighty percent of castor oil is ricinoleic acid, a fatty
acid that binds to receptors located on the smooth
muscle cells of the intestines, once locked onto those
receptors, ricinoleic acid causes powerful contractions,
explaining castor oil’s reputation as a stimulant laxative
(Rouf, 2003). Castor oil is also reported to induce
diarrhea by increasing the volume of intestinal content by
prevention of the reabsorption of water. The liberation of
ricinoleic acid results in irritation and inflammation of the
intestinal mucosa, leading to release of prostaglandins,
which results in the stimulation of secretion, thereby,
preventing the reabsorption of sodium chloride and water.
Castor oil therefore incorporates both secretory and
motility diarrhea (Janssen, 2014). Inhibitors of
prostaglandin synthesis are known to delay diarrhea
induced with castor oil (Williamson, 1996)).
Loperamide is an opioid-receptor agonist that acts on
the µ-opioid receptors in the mesenteric plexus of the
large intestine by decreasing the activity of the
mesenteric plexus, which in turn decreases the tone of
the longitudinal and circular smooth muscles of the
intestinal wall.
This increases the amount of time
substances stay in the intestine.
The result of castor oil induced diarrhea studies
showed that the methanol leaf extract has comparable
inhibitory potentials on diarrhea with the antidiarrheal
drug, loperamide. The observed antidiarrheal effect of
extract may be linked to the same mechanism as of
loperamide action or possibly inhibition of prostaglandin
biosynthesis.
In the motility studies using charcoal meal, the extract
did not show significant percentage inhibition. This may
suggest that its antidiarrheal effects of the extract may be
due to other mechanisms outside the anti-motility.
However, this contrasts its behavior in test for
spasmolytic activity using guinea pig ileum where it
showed significant inhibition of smooth muscle
contractions produced by acetylcholine antagonist like
atropine.
The spasmolytic activity of methanol extract of leaves
of Lophira lanceolata was tested using smooth muscle
preparations. Acetylcholine is a neurotransmitter released
by the parasympathetic nervous system. It mediates its
action in the gut by stimulation of M3 receptors, hence,
regulating the peristaltic movement of the gut. Muscarinic
antagonists like atropine inhibits action of acetylcholine
thus leading to decrease in gut peristalsis.
Although the result of the studies showed that the
extract has 500 times and 20,000 times less activity than
atropine and promethazine respectively, it antagonized
the activity of acetylcholine more. This further suggests
the possibility of inhibition of smooth muscle via
muscarinic antagonistic activity like atropine.
CONCLUSION
The leaf extract of Lophira lanceolata possess
antidiarrheal and some spasmolytic effects. These
findings provide a scientific proof for the use of this plant
in the treatment of diarrhea in humans and livestock.
Further studies are required to isolate and characterize
the exact active constituents of the leaf extract
responsible for its anti-diarrhea activity.
ACKNOWLEDGEMENT
The authors wish to thank Mr. A Ozioko, a Taxonomist
and staff of the International Centre for Ethno medicine
and Drug Development (InterCEDD) Nsukka, Nigeria for
the botanical identification and collection of the plant.
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